Matthias J. Feige

An Unfolded CH1 Domain Controls the Assembly and Secretion of IgG Antibodies

A prerequisite for antibody secretion and function is their assembly into a defined quaternary structure, composed of two heavy and two light chains for IgG. Unassembled heavy chains are actively retained in the endoplasmic reticulum (ER). Here, we show that the C(H)1 domain of the heavy chain is intrinsically disordered in vitro, which sets it apart from other antibody domains. It folds only upon interaction with the light-chain C(L) domain. Structure formation proceeds via a trapped intermediate and can be accelerated by the ER-specific peptidyl-prolyl isomerase cyclophilin B. The molecular chaperone BiP recognizes incompletely folded states of the C(H)1 domain and competes for binding to the C(L) domain. In vivo experiments demonstrate that requirements identified for folding the C(H)1 domain in vitro, including association with a folded C(L) domain and isomerization of a conserved proline residue, are essential for antibody assembly and secretion in the cell.

Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions

The endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain–binding protein), together with cochaperones, has important roles in all of these processes. The functional cycle of Hsp70s is determined by conformational transitions that are required for substrate binding and release. Here, we used the intrinsically disordered CH1 domain of antibodies as an authentic substrate protein and analyzed the conformational cycle of BiP by single-molecule and ensemble Förster resonance energy transfer (FRET) measurements. Nucleotide binding resulted in concerted domain movements of BiP. Conformational transitions of the lid domain allowed BiP to discriminate between peptide and protein substrates. A major BiP cochaperone in antibody folding, ERdj3, modulated the conformational space of BiP in a nucleotide-dependent manner, placing the lid subdomain in an open, protein-accepting state.

Disulfide bonds in ER protein folding and homeostasis

Proteins that are expressed outside the cell must be synthesized, folded, and assembled in a way that ensures they can function in their designate location. Accordingly, these proteins are primarily synthesized in the endoplasmic reticulum (ER), which has developed a chemical environment more similar to that outside the cell. This organelle is equipped with a variety of molecular chaperones and folding enzymes that both assist the folding process, while at the same time exerting tight quality control measures that are largely absent outside the cell. A major post-translational modification of ER-synthesized proteins is disulfide bridge formation, which is catalyzed by the family of protein disulfide isomerases. As this covalent modification provides unique structural advantages to extracellular proteins, multiple pathways to disulfide bond formation have evolved. However, the advantages that disulfide bonds impart to these proteins come at a high cost to the cell. Very recent reports have shed light on how the cell can deal with or even exploit the side reactions of disulfide bond formation to maintain homeostasis of the ER and its folding machinery.

BiP and Its Nucleotide Exchange Factors Grp170 and Sil1: Mechanisms of Action and Biological Functions

BiP (immunoglobulin heavy-chain binding protein) is the endoplasmic reticulum (ER) orthologue of the Hsp70 family of molecular chaperones and is intricately involved in most functions of this organelle through its interactions with a variety of substrates and regulatory proteins. Like all Hsp70 family members, the ability of BiP to bind and release unfolded proteins is tightly regulated by a cycle of ATP binding, hydrolysis, and nucleotide exchange. As a characteristic of the Hsp70 family, multiple DnaJ-like co-factors can target substrates to BiP and stimulate its ATPase activity to stabilize the binding of BiP to substrates. However, only in the past decade have nucleotide exchange factors for BiP been identified, which has shed light not only on the mechanism of BiP-assisted folding in the ER but also on Hsp70 family members that reside throughout the cell. We will review the current understanding of the ATPase cycle of BiP in the unique environment of the ER and how it is regulated by the nucleotide exchange factors, Grp170 (glucose-regulated protein of 170kDa) and Sil1, both of which perform unanticipated roles in various biological functions and disease states.

Quality Control of Integral Membrane Proteins by Assembly-Dependent Membrane Integration

Cell-surface multiprotein complexes are synthesized in the endoplasmic reticulum (ER), where they undergo cotranslational membrane integration and assembly. The quality control mechanisms that oversee these processes remain poorly understood. We show that less hydrophobic transmembrane (TM) regions derived from several single-pass TM proteins can enter the ER lumen completely. Once mislocalized, they are recognized by the Hsp70 chaperone BiP. In a detailed analysis for one of these proteins, the αβT cell receptor (αβTCR), we show that unassembled ER-lumenal subunits are rapidly degraded, whereas specific subunit interactions en route to the native receptor promote membrane integration of the less hydrophobic TM segments, thereby stabilizing the protein. For the TCR α chain, both complete ER import and subunit assembly depend on the same pivotal residue in its TM region. Thus, membrane integration linked to protein assembly allows cellular quality control of membrane proteins and connects the lumenal ER chaperone machinery to membrane protein biogenesis.

The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity.

Folding intermediates play a key role in defining protein folding and assembly pathways as well as those of misfolding and aggregation. Yet, due to their transient nature, they are poorly accessible to high-resolution techniques. Here, we made use of the intrinsically slow folding reaction of an antibody domain to characterize its major folding intermediate in detail. Furthermore, by a single point mutation we were able to trap the intermediate in equilibrium and characterize it at atomic resolution. The intermediate exhibits the basic β-barrel topology, yet some strands are distorted. Surprisingly, two short strand-connecting helices conserved in constant antibody domains assume their completely native structure already in the intermediate, thus providing a scaffold for adjacent strands. By transplanting these helical elements into β2-microglobulin, a highly homologous member of the same superfamily, we drastically reduced its amyloidogenicity. Thus, minor structural differences in an intermediate can shape the folding landscape decisively to favor either folding or misfolding.

C-terminal mutations destabilize SIL1/BAP and can cause Marinesco-Sjögren syndrome.

Background: Mutations in SIL1 can cause Marinesco-Sjögren syndrome. Results: Deletion or mutation of the last 5–6 amino acids destabilized SIL1. Conclusion: KELR does not represent a divergent ER retention sequence but is required for structural stability of the protein. Significance: These new insights might be a first step toward a possible pharmacological treatment of certain types of MSS by specifically stabilizing the mutant SIL1 protein. Marinesco-Sjögren syndrome (MSS) is an autosomal recessive, neurodegenerative, multisystem disorder characterized by severe phenotypes developing in infancy. Recently, mutations in the endoplasmic reticulum (ER)-associated co-chaperone SIL1/BAP were identified to be the major cause of MSS. SIL1 acts as a nucleotide exchange factor for BiP, the ER Hsp70 orthologue, which plays an essential role in the folding and assembly of nascent polypeptide chains in the ER. SIL1 facilitates the release of BiP from unfolded protein substrates, enabling the subsequent folding and transport of the protein. Although most mutations leading to MSS result in deletion of the majority of the protein, three separate mutations have been identified that disrupt only the last five or six amino acids of the protein, which were assumed to encode a divergent ER retention motif. This study presents an in depth analysis of two of these mutants and reveals that the phenotype in the affected individuals is not likely to be due to depletion of SIL1 from the ER via secretion. Instead, our analyses show that the mutant proteins are particularly unstable and either form large aggregates in the ER or are rapidly degraded via the proteasome. In agreement with our findings, homology modeling suggests that the very C-terminal residues of SIL1 play a role in its structural integrity rather than its localization. These new insights might be a first step toward a possible pharmacological treatment of certain types of MSS by specifically stabilizing the mutant SIL1 protein.

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins

Significance Sharks are among the evolutionary oldest living organisms with an immune system that possesses a number of elements similar to ours, including antibodies. In this article, we present structural insights into one of the most ancient antibodies, shedding light on the molecular evolution of the immune system and the structural features of heavy chain-only antibodies. Sharks enrich urea in their blood to prevent osmotic loss of water in the marine environment. Urea, however, denatures proteins if they are not sufficiently stable. Indeed, we find that shark antibodies are particularly stable. We pinpointed specific features responsible for their high stability and found that transplanting them into a human antibody increased its secretion. Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules.

Conformational selection in substrate recognition by Hsp70 chaperones.

Hsp70s are molecular chaperones involved in the folding and assembly of proteins. They recognize hydrophobic amino acid stretches in their substrate binding groove. However, a detailed understanding of substrate specificity is still missing. Here, we use the endoplasmic reticulum-resident Hsp70 BiP to identify binding sites in a natural client protein. Two sites are mutually recognized and form stable Hsp70-substrate complexes. In silico and in vitro analyses revealed an extended substrate conformation as a crucial factor for interaction and show an unexpected plasticity of the substrate binding groove. The basic binding mechanism is conserved among different Hsp70s.

Rate of Loop Formation in Peptides: A Simulation Study

Experimental techniques with high temporal and spatial resolution extend our knowledge of how biological macromolecules self-organise and function. Here, we provide an illustration of the convergence between simulation and experiment made possible by techniques such as triplet-triplet energy transfer and fluorescence quenching with long-lifetime and fast-quenching fluorescent probes. These techniques have recently been used to determine the average time needed for two residues in a peptide or protein segment to form a contact. The timescale of this process is accessible to computer simulation, providing a microscopic interpretation of the data and yielding new insight into the disordered state of proteins. Conversely, such experimental data also provide a test of the validity of alternative choices for the molecular models used in simulations, indicating their possible deficiencies. We carried out simulations of peptides of various composition and length using several models. End-to-end contact formation rates and their dependence on peptide length agree with experimental estimates for some sequences and some force fields but not for others. The deviations are due to artefactual structuring of some peptides, which is not observed when an atomistic model for the solvation water is used. Simulations show that the observed experimental rates are compatible with considerably different distributions of the end-to-end distance; for realistic models, these are never Gaussian but indicative of a rugged energy landscape.